The mitochondrial KATP channel (mitoKATP) is an inner membrane protein whose function is to regulate matrix volume and, thereby, to preserve the architecture of the intermembrane space. MitoKATP has been postulated to be the end-effector of ischemic cardioprotection. The long-term goal of this project is to understand the mechanism and regulation of mitoKATP and, of particular relevance to this FIRCA project, to understand its subunit structure-function. MitoKATP is hypothesized to consist of two subunits, a K+ channel (mitoKIR) and a regulatory sulfonylurea receptor (mitoSUR). This project exploits our ability to isolate mitoKIR, without mitoSUR, and to study its electrical properties in lipid bilayer membranes (BLM). The specific aims are to elucidate the electrical properties of mitoKIR; to characterize regulation of mitoKIR and compare with the intact mitoKATP; and to purify sufficient mitoKIR for peptide microsequencing. The channel protein will be isolated from mitochondria by a novel ethanol/water extraction method, and the highly purified mitoKIR will be reconstituted into BLM for electrophysiological studies and also used for microsequencing.